Comparison of fluorescent labels for oligosaccharides and introduction of a new postlabeling purification method.
Labeling of oligosaccharides with fluorescent dyes is the prerequisite for their sensitive analysis by high-performance liquid chromatography (HPLC). In this work, we present a fast new postlabeling cleanup procedure that requires no device other than the reaction vial itself. The procedure can be applied to essentially all labeling reagents. We also compare the performance of 15 different labels for N-glycan analysis in various analytical procedures. We took special care to prevent obscuring influences from incomplete derivatization and signal quenching by impurities. Procainamide emerged as more sensitive than anthranilic acid for normal-phase HPLC, but its chromatographic performance was not convincing. 2-aminopyridine was the label with the lowest retention on reversed-phase and graphitic carbon columns and, thus, appears to be most suitable for glycan fractionation by multidimensional HPLC. Most glycan derivatives performed better than native sugars in matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) and electrospray ionization-MS (ESI-MS), but the gain was small and hardly sufficient to compensate for sample loss during preparation.
Pabst
Martin
M
Department of Chemistry, University of Natural Resources and Applied Life Sciences (BOKU), 1190 Vienna, Austria.
Kolarich
Daniel
D
Pöltl
Gerald
G
Dalik
Thomas
T
Lubec
Gert
G
Hofinger
Andreas
A
Altmann
Friedrich
F
D000096:Acetone / Q000737:chemistry; D000586:Amination; D002851:Chromatography, High Pressure Liquid; D005456:Fluorescent Dyes / Q000737:chemistry*; D009844:Oligosaccharides / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification*; D011134:Polysaccharides / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D005456:Fluorescent Dyes; D009844:Oligosaccharides; D011134:Polysaccharides; D000096:Acetone